Use of a plant oil product as an agent for increasing the synthesis of skin lipids

ABSTRACT

The present invention relates to the use of a plant oil product as an agent for increasing the synthesis of skin lipids, especially the lipids of the epidermal skin barrier, in or for the preparation of a cosmetic, pharmaceutical or dermatological composition. The invention also relates to a method for cosmetic treatment with a cosmetic, pharmaceutical or dermatological composition for increasing the synthesis of skin lipids, especially the lipids of the epidermal skin barrier, and to the use of the plant product as a food additive.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/088,851, filed on Mar. 21, 2002, the disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the use of a plant oil product as anagent for increasing the synthesis of skin lipids, especially the lipidsof the epidermal skin barrier, in or for the preparation of a cosmetic,pharmaceutical or dermatological composition. The invention also relatesto a method for cosmetic treatment with a cosmetic, pharmaceutical ordermatological composition for increasing the synthesis of skin lipids,especially the lipids of the epidermal skin barrier, and to the use ofthe plant product as a food additive.

The skin consists mainly of three layers: the epidermis, the dermis andthe hypodermis.

The outermost layer, the epidermis, is characterized by organizationinto strata corresponding to a state of increasing differentiation ofthe keratinocytes, from the deepest region (stratum basale) to theoutermost region (stratum corneum) within which anuclear elements(corneocytes) are included in a multilamellar extracellular lipidstructure, the intercorneocytic cement, responsible for the skin's waterbarrier function and for protection against external attack.

The lamellar bodies, or Oddland bodies, secreted by the stratumgranulosum, a layer intermediate between the stratum basale and thestratum corneum, contain cholesterol, phospholipids andglucosylceramides and also selective hydrolases. These enzymes convertphospholipids and glucosylceramides into free fatty acids and ceramideswhich form, with cholesterol and cholesteryl sulfate, the intercellularlamellar bilayers of the stratum corneum. Ceramides participatepredominantly in the formation of the barrier formed by the stratumcorneum and in regulating water flow by unifying the lamellae. A largereduction in the content of/and the type of ceramide is especiallyobserved in atopic dermatitis (or atopic eczema) or in acne (ceramide 1)and in dry skin and pruritus in the elderly. Cholesteryl sulfate, via aspecific sulfatase, is in equilibrium with cholesterol(leaflet-fluidizing agent). They play an important role in corneocytecohesion and thus in skin desquamation, and also in skin comfort.

An impairment in this skin barrier caused by external attack (UVradiation, wind, cold, detergents, etc.), by the natural and inexorablephenomenon of aging and/or by pathological or nonpathologicaldysfunctions (sensitive, irritated or reactive skin) is manifested in adisruption in the epidermal homeostasis which it is desirable to able toprevent and/or treat both cosmetically and pharmaceutically andespecially dermatologically.

For example, the article by Ruby Ghadially et al., “Decreased EpidermalLipid Synthesis Accounts for Altered Barrier Function in Aged Mice”, TheJournal of Investigate Dermatology, vol. 106, no. 5, May 1996, teachesthat an impaired skin barrier and also an abnormal content of lipids inan aged mouse epidermis can be explained by an impaired synthesis of theepidermal lipids.

DETAILED DESCRIPTION

There is thus a need to be able to stimulate the synthesis of the skinlipids, especially the lipids of the epidermal skin barrier, so as to beable in particular to restore the skin barrier function of the epidermisand/or to combat various skin disorders associated with a reduction inthe synthesis of skin lipids, especially the lipids of the epidermalskin barrier.

It has now been found, entirely surprisingly and unexpectedly, that theuse of certain plant oil products makes it possible advantageously tosynthesize skin lipids, especially lipids of the epidermal skin barrier.

One subject of the present invention is thus the use of at least oneplant oil product chosen from the group consisting of oil distillates ofplant oil, unsaponifiable materials from plant oil, furan lipids ofplant oil and mixtures thereof as agents for increasing the synthesis ofskin lipids, especially the lipids of the epidermal skin barrier knownto those skilled in the art, like those mentioned above, in or for thepreparation of a composition containing a cosmetically, pharmaceuticallyor dermatologically acceptable medium.

In particular, the use according to the invention is characterized inthat the skin lipids are chosen, inter alia, from the epidermal lipidsof the group consisting of cholesterol, cholesteryl sulfate, ceramides 1and 2 and mixtures thereof.

Among the plant oils that may be used, mention may be made in particularof sunflower oil, palm oil, palm kernel oil, coconut oil, grapeseed oil,black mustard oil, poppyseed oil, karite butter oil, sweet almond oil,soybean oil, avocado oil, groundnut oil, cotton oil, sesame oil, oliveoil, corn oil, cocoa bean oil, castor oil, behen oil, flax oil, rapeseedoil, annatto oil, wheatgerm oil, safflower oil, walnut oil, hazelnut oiland turnipseed oil.

According to the invention, the expression “oil distillate of a plantoil” means a plant oil which has been subjected to a step ofconcentration of its unsaponifiable material.

The unsaponifiable material is the fraction of a fatty substance which,after prolonged action of an alkaline base, remains insoluble in waterand may be extracted with an organic solvent. Five major groups ofsubstances are present in most unsaponifiable materials of plant oils:saturated or unsaturated hydrocarbons, aliphatic or terpenic alcohols,sterols, phytosterols, tocopherols and carotenoid and xanthophyllpigments.

Plant oils whose unsaponifiable material and/or oil distillate are richin tocopherols and/or phytosterols are particularly preferred for useaccording to the invention. A person skilled in the art readilyunderstands that the term “rich” refers to tocopherol and phytosterolcontents that are respectively above the mean respective contentsobtained on consideration of all the plant oils known to those skilledin the art and in particular those mentioned above.

Various methods may be used to concentrate the unsaponifiable materialof a plant oil: crystallization under cold conditions, liquid-liquidextraction, molecular distillation.

Molecular distillation is particularly preferred, and is preferablycarried out at a temperature of between about 180° C. and about 280° C.,maintaining a pressure of between about 10⁻³ mmHg and about 10⁻² mmHgand preferably of the order of 10⁻³ mmHg. The concentration ofunsaponifiable material in the distillate may be up to 60%.

This molecular distillation, as well as any other molecular distillationfor the preparation of the plant oil products to be used according tothe invention, as described below, is preferably carried out using adevice chosen from molecular distillation equipment of centrifugal typeand molecular devices of scraped-film type.

Molecular distillation equipment of centrifugal type is known to thoseskilled in the art. For example, patent application EP-0 493 144discloses a molecular distillation device of this type. In general, theproduct to be distilled is spread in a thin film over the heated surface(hot surface) of a conical rotor rotating at high speed. Thedistillation chamber is placed under vacuum. Under these conditions,evaporation, rather than boiling, takes place, from the hot surface, ofthe constituents of the unsaponifiable material, the advantage beingthat the oil and the unsaponifiable material (these products beingnotoriously fragile) are not degraded during the evaporation.

Molecular distillation devices of scraped-film type, which are alsoknown to those skilled in the art, comprise a distillation chamber whichhas a rotating scraper, allowing the product to be distilled to bespread continuously over the evaporation surface (hot surface). Theproduct's vapours are condensed by means of a cold finger placed in thecentre of the distillation chamber. The peripheral supply and vacuumsystems are very similar to those of a centrifugal distillation device(supply pumps, vane vacuum pumps and oil diffusion pumps, etc.). Theresidues and distillates are recovered in glass round-bottomed flasks bygravitational flow.

According to one particularly preferred embodiment of the presentinvention, an oil distillate of sunflower oil is used.

Preferably, the oil distillate of sunflower oil is obtained by moleculardistillation of a food-grade sunflower oil. The distillation conditionsare preferably as follows:

-   -   temperature from 230 to 250° C.;    -   pressure from 10⁻³ to 10⁻² mmHg;    -   degree of distillation of about 5 to 10% by mass.

The degree of distillation may be defined as follows: it is the massratio, relative to 100%, of the mass of the distillate to the sum (massof the distillate+mass of the residue).

The distillate thus obtained, i.e. the oil distillate of sunflower oil,has an unsaponifiable material content of between about 6% and about 10%by weight, the remaining part being composed of sunflower oiltriglycerides.

The unsaponifiable material of a plant oil which may be used accordingto the invention is preferably chosen from the group consisting of theunsaponifiable material of avocado oil and the unsaponifiable materialof soybean oil, and mixtures thereof.

Comparison of the unsaponifiable material contents of various plantoils—soybean, cotton, coconut, olive and avocado—shows a very largeamount of unsaponifiable material in avocado oil, obtained by extractionaccording to various known processes. Typically, the contents obtainedrange from 2 to 7% of unsaponifiable material in avocado oil, comparedwith 0.5% in coconut oil, 1% in soybean oil and 1% in olive oil.

The higher content of unsaponifiable material in avocado oil comparedwith the other plant oils such as mentioned above is explained inparticular by the presence, in the unsaponifiable material of avocadooil, of constituents which are not generally found in the unsaponifiablematerial of many other plant oils, such as furan compounds andpolyhydroxylated fatty alcohols, and which, by themselves, representmore than 50% of the unsaponifiable material. The products specific tothis unsaponifiable material of avocado oil may be divided into twochemical fractions referred to as “fraction I” and “fraction H”. Theactive compounds for the use according to the invention are present inthe H fraction and its precursors. The H fraction appears first on a gaschromatograph of the unsaponifiable material of avocado oil.

As regards the unsaponifiable material of soybean oil, it may be notedthat this unsaponifiable material is mainly composed of sterols (40 to65%) and of tocopherols (≧10%). The main sterols are β-sitosterol (40 to70% of the total sterols), campesterol (15 to 30% of the total sterols)and stigmasterol (10 to 25% of the total sterols). The tocopherols arepresent in the form of a mixture of α-tocopherol (5 to 35% of totaltocopherols), γ-tocopherol (45 to 70% of the total tocopherols) andδ-tocopherol (10 to 43% of the total tocopherols).

Several processes have been described in the prior art to extract theunsaponifiable material of a plant oil.

Mention may be made in particular of the process for preparing anunsaponifiable material of avocado oil as described and claimed inpatent FR-2 678 632 in the name of Laboratoires Pharmascience. Thisprocess makes it possible to obtain an unsaponifiable material ofavocado oil which are rich in H fraction, compared with the conventionalprocesses for preparing the unsaponifiable material of avocado.

Thus, the unsaponifiable material of avocado oil used according to theinvention may be obtained from the fresh fruit, but, preferably, theunsaponifiable material of avocado is prepared from fruit that has beenheat-treated beforehand, prior to extracting the oil and saponifying it,as described in patent FR-2 678 632.

This heat treatment consists of a controlled drying of the fruit, whichis preferably fresh, for at least four hours, advantageously at least 10hours, preferably between about 24 and about 48 hours, at a temperaturepreferably of at least about 80° C. and preferably between about 80° C.and about 120° C.

Mention may also be made of the process for preparing the unsaponifiablematerial of soybean oil, obtained from a concentrate of unsaponifiablematerial of soybean oil. The said concentrate of unsaponifiable materialis prepared by molecular distillation according to a process such as theone described for lupin oil in patent application FR-A 762 512, butadapted to soybean oil. In this process, the soybean oil is distilled ina molecular distillation device of centrifugal type or of scraped-filmtype, at a temperature of between about 210° C. and 250° C. and under ahigh vacuum of between 0.01 and 0.001 millimeters of mercury (i.e. 0.13to 1.3 Pa). The distillate obtained has an unsaponifiable materialcontent of between 5% and 30% by weight and thus constitutes aconcentrate of unsaponifiable material of soybean oil. This concentrateis then saponified according to a conventional saponification process,in the presence of ethanolic potassium hydroxide. The mixture obtainedis extracted with dichloroethane in a counter current column. Finally,the solvent is removed from the solvent phase by passing it through afalling-film evaporator in order to recover the unsaponifiable materialof soybean.

According to one preferred embodiment of the present invention, theunsaponifiable material of a plant oil is a mixture of unsaponifiablematerial of avocado oil and of soybean oil, the weight ratio ofunsaponifiable material of avocado oil to the unsaponifiable material ofsoybean oil being between about 0.1 and about 9 and preferably betweenabout 0.25 and about 0.6.

In particular, it is advantageously possible to use the mixture ofunsaponifiable materials of avocado oil and of soybean oil as sold bythe company Laboratoires Pharmascience under the name “Piascledine 300®”which consists of a mixture of 33.3% by weight of unsaponifiablematerial of avocado and 66.6% by weight of unsaponifiable material ofsoybean, relative to the total weight of the mixture (the remaining 0.1%consisting of colloidal silica and butylhydroxytoluene).

According to the invention, the expression “furan lipids of a plant oil”means compounds comprising a linear C11-C19 hydrocarbon-based mainchain, which is saturated or comprises one or more ethylenic oracetylenic unsaturations, and a 2-furyl group at one of its ends. Amongthe furan lipids of a plant oil which may be used according to theinvention, the ones most particularly preferred are the furan lipids ofavocado. This is because avocado comprises specific lipids of furantype, the main component of which is a linoleic furan:

The furan derivatives of avocado oil have been described in particularin Farines, M. et al, 1995, J. of Am. Oil Chem. Soc. 72, 473. It isnowadays well established that the presence of these furan compounds inthe leaves or fruit depends not only on the variety (the varieties Hassand Fuerte being richest in furan compounds) but also on the method forobtaining the oil or another plant extract of avocado (hexane orethanolic extract of avocado leaves).

Specifically, it is known that these furan lipids are metabolites ofcompounds that are initially present in the fruit and leaves and which,under the effect of heat, dehydrate and cyclize to give furanderivatives.

For example, linoleic furan is derived from the thermal conversion ofthe following precursor:

According to the invention, the expression “furan lipids of avocado” inparticular means the components corresponding to the formula:

in which R is a linear C₁₁-C₁₉, preferably C₁₃-C₁₇, hydrocarbon-basedchain which is saturated or comprises one or more ethylenic oracetylenic unsaturations.

The known processes for obtaining these specific compounds from avocadofruit or from avocado fruit oil come down to either preparativechromatography or industrial processes for obtaining these furan lipidsas a mixture with the other unsaponifiable compounds of avocado, with amaximum content of furan-containing lipids which is at best between 50%and about 65% by weight only.

A novel process for preparing these furan lipids of avocado was thesubject of a patent application filed this very day. It consistsessentially of a process for selectively extracting the furan lipids ofavocado, characterized in that it comprises the steps consisting inpreparing an unsaponifiable material of avocado and then in subjectingthe unsaponifiable material of avocado to a step of moleculardistillation using temperature means regulated for a temperature ofbetween 100° C. and 160° C. and pressure means regulated for a pressureof between 10-3 mmHg and 5×10-2 mmHg.

This molecular distillation step using specific temperature and pressureconditions constitutes an essential characteristic of this process, incombination with the prior step of preparing the unsaponifiable materialalready described above.

According to the invention, the plant oil product as described above isused in a proportion of between about 0.01% and 100% by weight andpreferably between about 0.5% and about 10% by weight relative to thetotal weight of the composition.

The cosmetically, pharmaceutically or dermatologically acceptable mediumfor the use according to the invention may be any medium that issuitable for the presentation forms known to those skilled in the art,for topical, oral, enteral or parenteral administration.

In particular, this medium may be an oily solution, a water-in-oilemulsion, an oil-in-water emulsion, a microemulsion, an oily gel, ananhydrous gel, or a dispersion of vesicles, microcapsules ormicroparticles.

The composition for the use according to the invention is preferablysuitable for administration by topical application.

The advantageous effect of increasing the synthesis of the skin lipids,especially the lipids of the epidermal skin barrier, makes it possibleto prevent and/or treat, in other words allows the treatment of,impairments of the skin barrier formed mainly by the epidermal layers ofthe stratum corneum and the stratum granulosum as explained above.

Thus, the use according to the invention is characterized in that thecomposition is intended for treating dry skin and skin that has beensubjected to actinic radiation, especially UV radiation, such as solarradiation or radiation from a UV lamp, for example during an artificialtanning session.

The use according to the invention is also characterized in that thecomposition is intended for treating ichthyosis, acne, xerosis, atopicdermatitis (or atopic eczema), skin disorders associated with areduction in the content of skin lipids, especially the lipids of theepidermal skin barrier, and disorders of corneocyte cohesion and ofdesquamation of the skin, sensitive, irritated and reactive skin, andpruritus.

A subject of the present invention is also a method for cosmetictreatment disorders associated with aging of the skin, neighboringmucous membranes and/or integuments, characterized in that a compositioncontaining at least one plant oil product in a cosmetically acceptablemedium as described above is applied to the skin, neighboring mucousmembranes and/or integuments.

A subject of the invention is also a method for cosmetic treatment ofdisorders associated with drying of the skin, neighboring mucousmembranes and/or integuments, characterized in that a compositioncontaining at least one plant oil product in a cosmetically acceptablemedium as described above is applied to the skin, neighboring mucousmembranes and/or integuments.

A subject of the invention is also a method for cosmetic treatment ofdisorders of the skin, neighboring mucous membranes and/or integuments,resulting from an exposure to actinic radiation, especially UVradiation, characterized in that a composition containing at least oneplant oil product in a cosmetically acceptable medium as described aboveis applied to the skin and/or integuments.

According to one preferred embodiment of these cosmetic treatmentmethods, the plant oil product is present in the composition in aproportion of between about 0.01% and 100% by weight and preferablybetween about 0.5% and about 10% by weight relative to the total weightof the composition.

A subject of the present invention is also a cosmetic, pharmaceutical ordermatological composition for increasing the synthesis of skin lipids,especially the lipids of the epidermal skin barrier, characterized inthat it comprises at least one plant oil product in a cosmetically,pharmaceutically or dermatologically acceptable medium, as describedabove.

Preferably, this composition is characterized in that the plant oilproduct is present in the composition in a proportion of between about0.01% and 100% by weight and preferably between about 0.5% and about 10%by weight relative to the total weight of the composition.

Finally, another subject of the invention is the use of at least oneplant oil product, as described above, as an additive in a food forhumans and/or animals.

This food use is preferably characterized in that the plant oil productis present in the food in a proportion of between about 0.1% and about20% by weight relative to the total weight of the food.

The examples which follow are intended to illustrate the presentinvention and should not in any way be interpreted as possibly limitingits scope.

Unless otherwise specified, the percentages indicated in the examplesbelow are percentages by weight.

Example 1 Preparation of Plant Oil Products and their Use According tothe Invention in the Form of Oil-in-Water Emulsions

The following compositions 1.1 to 1.3 and the placebo composition wereeach prepared in the following way:

The constituent components of the aqueous phase (water and glycerol) areplaced in a water bath at 75° C. The components of the fatty phase,except for the Sepigel 305, the Silicone SF 1202 and the plant oilproduct (prepared below, respectively, under the names “sunflower oildistillate 1”, “unsaponifiable materials 1” and “lipids 1”), are placedin a water bath at 75° C. Just before performing the emulsification, theSilicone 1202 and the respective plant oil product are added to thefatty phase. The emulsion is then prepared by turbomixing at low speedby incorporating the fatty phase into the aqueous phase. When thepreparation has reached a temperature of 60° C., the Sepigel 305 isadded with high-speed turbomixing. The composition is then allowed tocool at rest to room temperature, before being used in the testsdescribed below.

1.1) Composition 1.1: Use of an Oil Distillate of Sunflower Oil

A sunflower oil distillate is prepared by molecular distillation, in amolecular distillation machine of centrifugal type, of a commercialfood-grade sunflower oil. The distillation conditions are as follows:

-   -   temperature 220° C.;    -   pressure of 10⁻³ mmHg;    -   degree of distillation: 6/7% by mass    -   feed rate: 18 kg/h

The distillate obtained, sunflower oil distillate, has a content ofunsaponifiable materials of about 6.2% by weight, the remaining portionbeing composed of sunflower oil triglycerides. The oil distillate thusobtained is referred to as “sunflower oil distillate 1”.

Composition 1.1 % (INCI formula) (by weight) Aqueous phase Water 67.3Glycerol 4 Fatty phase Sorbitan tristearate 1.85 PEG-40 stearate 3.15Silicone SF 1202 3 Cetiol Oe 1 Petroleum jelly codex 2.5 Glycerylstearate 6 Decyl pentanoate 3 Sunflower oil distillate 1 2 Beeswax 3PEG-2 stearate 1 C12-15 alcohol benzoate 1 Phenonip 0.7 Sepigel 305 0.3TOTAL 100%

1.2) Composition 1.2: Use of a Mixture of an Unsaponifiable Materialfrom Avocado and from Soybean

The mixture of unsaponifiable materials from avocado oil and fromsoybean oil as sold by the company Laboratoires Pharmascience under thename “Piascledine 300®”, which consists of a mixture of 33.3% by weightof unsaponifiable materials from avocado and 66.6% by weight ofunsaponifiable materials from soybean, relative to the total weight ofthe mixture (the remaining 0.1% consisting of colloidal silica andbutyl-hydroxytoluene) is used. This mixture is referred to hereinbelowas “unsaponifiable materials 1”.

Composition 1.2 (INCI names) Aqueous phase Water 67.3 Glycerol 4 Fattyphase Sorbitan tristearate 1.85 PEG-40 stearate 3.15 Silicone SF 1202 3Cetiol Oe 1 Petroleum jelly codex 2.5 Glyceryl stearate 6 Decylpentanoate 3 Unsaponifiables 1 2 Beeswax 3 PEG-2 stearate 1 C12-15alcohol benzoate 1 Phenonip 0.7 Sepigel 305 0.5 TOTAL 100%

1.3) Composition 1.3: Use of Furan-Containing Lipids of Avocado

An unsaponifiable material from avocado is prepared as described inpatent FR-2 678 632. Its composition is as follows:

polyhydroxylated fatty alcohols 24.3% furan-containing lipids 55.5%sterols 3.1% squalene 1.4% others 15.7% (1) (1) free fatty acids,hydrocarbons, tocopherols, fatty ketones and heavy pigments

This unsaponifiable material is subjected to molecular distillationusing the scraped-film molecular distillation machine sold by thecompany Leybold under the name “KDL4”. The distillation conditions areas follows:

-   -   hot-surface temperature: 108° C.    -   pressure: 10⁻³ mmHg    -   rotation speed of the shaft: 240 rpm    -   flow rate of unsaponifiable material from avocado: 400 ml/h

Yield of distillate: 48.6%

Composition of the Distillate:

polyhydroxylated fatty alcohols: n.m. furan-containing lipids 99.1%sterols n.m. squalene n.m. others 0.9% (1) (1) free fatty acids,hydrocarbons and fatty ketones (“n.m.”: not measurable, that is to say acontent of less than 0.05%)

This is thus a distillate that is very rich in furan-containing lipidssince the content of said lipids exceeds 99%. This distillate isreferred to hereinbelow as “lipids 1”

Composition 1.3 % (INCI names) (by weight) Aqueous phase Water 69Glycerol 4 Fatty phase Sorbitan tristearate 1.85 PEG-40 stearate 3.15Silicone SF 1202 3 Cetiol Oe 1 Petroleum jelly codex 2.5 Glycerylstearate 6 Decyl pentanoate 3 Lipids 1 0.3 Beeswax 3 PEG-2 stearate 1C12-15 alcohol benzoate 1 Phenonip 0.7 Sepigel 305 0.5 TOTAL 100%

1.4) Placebo Composition

Placebo composition % (INCI names) (by weight) Aqueous phase Water 69.3Glycerol 4 Fatty phase Sorbitan tristearate 1.85 PEG-40 stearate 3.15Silicone SF 1202 3 Cetiol Oe 1 Petroleum jelly codex 2.5 Glycerylstearate 6 Decyl pentanoate 3 Beeswax 3 PEG-2 stearate 1 C12-15 alcoholbenzoate 1 Phenonip 0.7 Sepigel 305 0.5 TOTAL 100%

Example 2 In Vitro Evaluation of the Effect of Compositions 1.1, 1.2,1.3 and Placebo on the Metabolism of the Epidermal Lipids in anOrganotypical Model of Whole Human Skin in Culture

The following abbreviations are used in the text hereinbelow:

EGF: epidermal growth factor; TLC: thin layer chromatography; MCF:culture medium for disks of human skin; MIF: incubation medium for disksof human skin; PBS: phosphate-buffered saline.

The object of this study is to study the effect of the four compositions1.1, 1.2, 1.3 and placebo described above on the metabolism of epidermallipids.

The study is performed in vitro in an organotypical model of whole humanskin in culture. Two techniques successively used:

-   -   measurement of the incorporation of acetate radiolabeled with        carbon 14 into the neosynthesized epidermal lipids in toto;    -   analysis by thin layer chromatography to separate the main        classes of neosynthesized radiolabeled epidermal lipids.

The effect of the test products is compared with that observed in thepresence of epidermal growth factor (EGF), diluted in the culture mediumfor the human skin disks, and in the presence of a commerciallyavailable cosmetic formulation containing lactic acid. EGF and lacticacid both stimulate, in a known manner, the synthesis of ceramides bythe keratinocytes (Ponec M. Gibbs S., Weerheim A., Kempenaar J., MulderA. and Mommaas A. M. —Epidermal growth factor and temperature regulatekeratinocytes differentiation—Arch. Dermatol. Res., 1997, 289, 317-326;and Rawlings A. V., Davies A., Carlomusto M., Pillai S. Ahang K.,Kosturbo R., Verdejo P., Feinberg C., Nguyen L. and Chandar P. —Effectof lactic acid isomers on keratinocyte ceramide synthesis, stratumcorneum lipid levels and stratum corneum barrier function—Arch.Dermatol. Res., 1996, 288, 383-390).

1) Materials and Method

1.1) Test Products, Reference Products and Reagents

Compositions 1.1, 1.2, 1.3 and placebo were prepared as described above.The EGF was obtained from R&D Systems. The cosmetic formulationcontaining lactic acid, referred to hereinbelow as “lactic acid”, waspurchased in the supermarket distribution network.

The solution for rinsing the human skin disks after incubation is PBSbuffer: 8 g/l NaCl; 1.15 g/l Na2HPO4; 0.2 g/l KH2PO4; 0.2 g/l KCl; 0.1g/l CaCl2; 0.1 g/l MgCl2; pH 7.4.

The other reagents, of analytical grade, are obtained from Carlo Erba,Gibco and Sigma, except where otherwise indicated.

1.2) Test System

A fragment of human skin was collected after an abdominal plasticsurgery operation. This was performed on a 24 year old woman (subject10129). Skin disks 8 mm in diameter were cut out using a sample punch.

The skin disks are placed in gondolas. The gondolas are placed inculture wells containing MCF medium, composed of MEM/M199 medium (3/4,1/4; v/v) supplemented with penicillin (50 IU/ml), streptomycin (500□g/ml), sodium bicarbonate (0.2%, w/v) and FCS (2%, v/v).

1.3) Incubation of the Test Products and the Reference Products with theTest System

The test products are tested undiluted. They are placed at the center ofeach human skin disk, in a proportion of 10 mg/cm2. The incubationmedium for the human skin disks (MIF medium) is composed of MCF mediumcontaining 1 μCi/ml of acetate labeled with carbon 14 (Amersham,specific activity: 57 mCi/mmol).

The EGF is tested at 10 ng/ml in the MIF medium. The lactic acid is usedin topical application (10 mg/cm2).

The human skin disks are incubated in the presence of the test productsand the reference products for 18 hours at 37° C. in a humid atmospherecontaining 5% CO₂.

Control skin disks are incubated in parallel in the presence of testproducts and reference products.

Each experimental condition is performed in quadruplicate.

The following time scale is used:

↑: topical application of the test products and the reference productsand dilution of the EGF in the incubation medium for the skin disks Δ:addition of acetate labeled with carbon 14 to the culture medium □:dermal/epidermal dissociation and evaluation of the effects

1.4) Evaluation of the Effects

1.4.1) Neosynthesis of the Total Epidermal Lipids

At the end of the incubation, the human skin disks are rinsed thoroughlywith PBS buffer. The epidermis of each skin disk is dissociated from thedermis by means of a controlled heat shock (MilliQ water, 2 min, 62°C.). The epidermises thus dissociated are digested with trypsin (ICN,1%, w/v) overnight at 37° C. The cell dissociation is facilitated by theaction of ultrasound.

The neosynthesized lipids, labeled with carbon 14, are extracted bypartition between an organic phase (1/4 methanol/chloroform, v/v) and anaqueous phase (0.25M potassium chloride). The organic phase isevaporated under nitrogen and the residues are taken up in a 2/1chloroform/methanol mixture (v/v).

The radioactivity of each sample, corresponding to the amount of acetateincorporated into the neosynthesized lipids, is measured by liquidscintillation.

The results are expressed in cpm/mg of epidermis.

1.4.2) Nature of the Neosynthesized Epidermal Lipids

Starting with the samples of extracted lipids, 20 μL aliquots,corresponding to 3 700 cpm, are placed on silica 60 (Merck)chromatography plates. These plates are developed in three successivesolvents:

-   -   38/2/10 chloroform/acetone/methanol (v/v/v),    -   40/5/5 chloroform/acetone/methanol (v/v/v),    -   36/10/3/1 chloroform/ethyl acetate/ether/methanol (v/v/v/v).

This system makes it possible to separate the cholesteryl sulfate, thecerebrosides, the ceramides, the cholesterol and the tri-+diglycerides.The most polar lipids, referred to hereinbelow as “polar lipids”, remainon the base line.

The silica plates are then placed in exposure with films forautoradiography for 15 days (Amersham, Hyperfilm beta max).

The position of the various classes of lipids—polar lipids, cholesterylsulfate, cerebrosides, ceramides 1 and 2, cholesterol andtri-+diglycerides—is determined using suitable standards.

The radioactivity of the spots separated and revealed by means ofautoradiography is counted using a thin-film argon-methane radioactivityanalyzer (Berthold). The results are expressed as percentages of theradioactivity of the total lipids neosynthesized and deposited on theTLC.

1.5) Treatment of the Data

The groups of data (control group and treated groups) are compared bymeans of a one-factor analysis of variance (Anova 1, p<0.05), followedby a Dunnett test.

2) Results

After incubating overnight in the presence of the human skin disks, thetest compositions and the reference compositions have no significanteffect on the neosynthesis of the total epidermal lipids (table 3.1). Onthe other hand, they appreciably modify the proportion of the variousepidermal lipids in this total:

-   -   EGF at 10 ng/ml reduced the neosynthesis of cholesteryl sulfate        by 46% and increased the neosynthesis of ceramide 2 by 55%        (table 3.2);    -   lactic acid increases the neosynthesis of the cerebrosides by a        factor of 1.59 (table 3.2);    -   composition 1.1 increases the respective neosynthesis of        ceramides 1 and 2 by a factor of 2.26 and 4.61, and the        neosynthesis of cholesterol by a factor of 5.04. It reduces the        neosynthesis of the tri- and diglycerides by 73% (table 3.3);    -   composition 1.2 increases the neosynthesis of cholesteryl        sulfate by a factor of 1.32, the respective neosynthesis of        ceramides 1 and 2 by a factor of 2.47 and 2.51, and the        neosynthesis of cholesterol by a factor of 4.62. It reduces the        neosynthesis of the tri- and diglycerides by 77% (table 3.2);    -   composition 1.3 increases the neosynthesis of cholesteryl        sulfate by a factor of 1.24, the respective neosynthesis of        ceramides 1 and 2 by a factor of 1.59 and 3.66, and the        neosynthesis of cholesterol by a factor of 4.14. It reduces the        neosynthesis of the tri- and diglycerides by 84% (table 3.2);    -   the placebo composition reduces the neosynthesis of the tri- and        diglycerides by 59% (table 3.3) without causing a significant        increase in the various epidermal lipids analyzed.

In conclusion, under the experimental conditions adopted, compositions1.1, 1.2, 1.3 and placebo have no significant effect on the neosynthesisof the total epidermal lipids.

On the other hand, they significantly modify the proportion of thevarious classes of epidermal lipids separated by thin layerchromatography in the total.

They reduce the neosynthesis of the tri- and diglycerides to the benefitof the other epidermal lipids.

Composition 1.1 increases the neosynthesis of cholesterol (withoutmodifying the neosynthesis of cholesteryl sulfate) and the neosynthesisof the ceramides.

Compositions 1.2 and 1.3 increase the neosynthesis of cholesterylsulfate and cholesterol, and the neosynthesis of the ceramides.

The placebo composition does not bring about a significant increase inthe various epidermal lipids analyzed.

3) Tables of Results

TABLE 3.1) Effect of compositions 1.1, 1.2, 1.3 and placebo and also ofEGF and of a cosmetic formulation containing lactic acid on theneosynthesis of the total epidermal lipids in disks of whole human skin,after incubation for 18 hours EGF Lactic Composition CompositionComposition Placebo Control 10 ng/ml acid 1.2 1.3 1.1 composition4183.35 3104.63 1864.31 2479.26 3646.42 873.10 1506.12 4407.89 5043.664919.12 3858.70 5255.70 3726.00 4154.88 3691.69 3606.76 4027.67 6571.133900.53 3802.63 4429.96 1062.72 4565.02 1076.21 2457.78 3209.16 905.552373.94 3336.41 +/− 4080.02 +/− 2971.83 +/− 3841.72 +/− 1934.04 4002.95+/− 882.63 2326.82 +/− 1660.22 3116.23 +/− 1408.09 1545.02 883.021800.62 100 122 89 115 120 70 93 The results are expressed in cpm/mg ofepidermis In bold: mean and standard deviation In italics: percentage ofthe control group *: mean significantly different from the control group(p < 0.05)

TABLE 3.2 Effect of compositions 1.2 and 1.3 and also of EGF and of acosmetic formulation containing lactic acid on the neosynthesis of thepolar lipids, of cholesteryl sulfate, of the cerebrosides, of ceramides1 and 2, of cholesterol and of the tri-+diglycerides in disks of wholehuman skin, after incubation for 18 hours Cholesteryl Tri-+di- ProductPolar lipids sulfate Cerebrosides Ceramide 1 Ceramide 2 Cholesterolglycerides Control 28.94 6.04 1.89 2.03 2.03 8.23 50.83 24.96 4.80 2.082.67 2.07 5.04 53.76 31.74 6.26 4.02 2.71 2.15 7.89 45.23 42.90 5.124.44 1.67 1.47 5.61 38.84 32.14 +/− 7.70  5.56 +/− 0.71 3.11 +/− 1.312.27 +/− 0.51  1.93 +/− 0.31 6.69 +/− 1.60 47.17 +/− 6.58 100 100 100100 100 100 100 EGF 10 ng/ml 57.27 2.31 2.87 2.02 3.81 7.07 42.67 34.423.69 3.82 1.41 2.38 9.46 44.82 27.69 3.44 4.77 3.41 2.74 7.86 50.0833.53 2.57 4.02 4.02 3.05 9.99 42.83  38.23 +/− 13.04 3.00* +/− 0.673.87 +/− 0.78 2.72 +/− 1.21 3.00* +/− 0.61 8.60 +/− 1.36 45.10 +/− 3.46119 54 125 120 155 128 96 Lactic acid 49.20 4.07 4.46 2.37 1.70 7.7733.43 33.04 4.16 4.54 2.64 3.32 9.72 42.58 64.49 5.90 6.07 2.94 2.578.07 31.95 53.51 4.25 4.68 2.58 1.44 8.67 24.86  50.06 +/− 13.05  4.60+/− 0.87 4.94* +/− 0.76  2.63 +/− 0.24  2.26 +/− 0.86 8.56 +/− 0.8633.21 +/− 7.28 156 83 159 116 117 128 70 Composition 24.57 6.89 4.412.78 6.86 44.35 10.13 1.2 28.71 8.34 5.91 5.94 3.34 35.04 12.72 33.096.86 4.02 4.55 4.79 36.97 9.72 35.78 7.35 7.76 9.20 4.37 34.01 11.5230.54 +/− 4.93 7.36* +/− 0.69 5.53 +/− 1.70 5.62* +/− 2.72  4.84* +/−1.48 37.59* +/− 4.67  11.02* +/− 1.37  95 132 178 247 251 562 23Composition 38.88 6.23 6.23 3.20 8.92 28.63 7.92 1.3 43.70 8.25 4.553.51 7.25 24.34 8.41 37.70 6.88 3.97 3.52 6.69 33.55 7.68 47.94 6.156.13 4.24 5.40 24.30 5.85 42.06 +/− 4.70 6.88* +/− 0.97 5.22 +/− 1.133.62* +/− 0.44  7.07* +/− 11.46 27.71* +/− 4.39  7.47* +/− 1.12 131 124168 159 366 414 16 The results are expressed as a percentage of theradiolabeled total lipids applied onto the TLC. In bold: mean andstandard deviation In italics: percentage of the control group +: meansignificantly different from the control group (p < 0.05)

TABLE 3.3 Effect of compositions 1.1 and placebo on the neosynthesis ofthe polar lipids, of cholesteryl sulfate, of the cerebrosides, ofceramides 1 and 2, of cholesterol and of tri-+ diglycerides in disks ofwhole human skin, after incubation for 18 hours Cholesteryl Tri-+Product Polar lipids sulfate Cerebrosides Ceramide 1 Ceramide 2Cholesterol diglycerides Control 28.94 6.04 1.89 2.03 2.03 8.23 50.8324.96 4.80 2.08 2.67 2.07 5.04 53.76 31.74 6.26 4.02 2.71 2.15 7.8945.23 42.90 5.12 4.44 1.67 1.47 5.61 38.84 32.14 +/− 7.70 5.56 +/− 0.713.11 +/− 1.31 2.27 +/− 0.51 1.93 +/− 0.31 6.69 +/− 1.60  47.17 +/− 6.58100 100 100 100 100 100 100 Composition 1.1 37.24 6.43 4.35 5.12 8.3832.19 16.28 32.48 4.83 5.45 4.22 7.77 32.51 12.74 31.92 3.81 5.68 5.438.06 32.23 10.86 25.87 4.41 4.49 5.75 11.37 38.00 10.11 31.88 +/− 4.664.87 +/− 1.12 4.99 +/− 0.67 5.13* +/− 0.66  8.90* +/− 1.67  33.73* +/−2.85  12.50* +/− 2.75 99 88 161 226 461 504 26 Placebo composition 73.679.27 7.69 2.44 2.05 3.03 18.60 58.70 11.03 7.60 2.51 3.39 3.71 13.0755.90 6.63 3.71 4.21 3.67 8.66 17.20 63.39 5.69 4.48 4.17 3.35 4.9327.98 62.92 +/− 7.81 8.16 +/− 2.44 5.87 +/− 2.07 3.33 +/− 0.99 3.12 +/−0.72 5.08 +/− 2.51 19.21* +/− 6.30 196 147 189 147 161 76 41 The resultsare expressed as a percentage of the radiolabeled total lipids appliedonto the TLC. In bold: mean and standard deviation In italics:percentage of the control group +: mean signficantly different from thecontrol group (p < 0.05)

Example 3 Composition of a Cream for Atopic Skin

INCI formulation % Water qs 100 Glycerol 15 Petrolatum 2 Hydrogenatedpalm kernel oil 5 Caprylic/capric triglycerides 5 Cyclomethicone 1Sucrose distearate 4 Dextrin 3 Sunflower (Helianthus annuus) seed oil 1unsaponifiables (1) Squalane 2 Candelilla (Euphorbia cerifera) wax 1Sucrose stearate 2 Oat (Avena sativa) flour 1 Dimethiconol 0.2Methylparaben 0.4 Propylparaben 0.3 Xanthan gum 0.2 Ceramide 3 0.2Total: 100% (1) Sunflower oil distillate 1 of example 1

Example 4 Composition of a Bath Oil for Atopic Skin

INCI formulation % Sunflower (Helianthus annulus) seed oil qs 100 Octylcocoate 15 Sweet almond (Prunus amygdalus dulcis) oil 15 Mineral oil 1PEG-6 isostearate 5 Sunflower (Helianthus annuus) seed oil 60unsaponifiables (1) Chamomile (Anthemis nobilis) oil 5 Propylene glycoldipelargonate 1 Lecithin 1 Laureth-2 0.5 Tocopherol 0.5 Ascorbylpalmitate 0.06 Total: 100% (1) Sunflower oil distillate 1 of example 1

Example 5 Clinical Study to Evaluate the Care Effect of Composition 1.1of Example 1 and Checking of its satisfactory local skin tolerance,Under Dermatological Control, after Single and Repeated Applications for4 Weeks, on an “Atopic” Adult Volunteer

1) Materials and Method

1.1 Aim of the Study

The aim is firstly to evaluate the care effect of a cosmetic product, byvarious biometrological measurements combined with clinical evaluations,and secondly to check their satisfactory local skin tolerance, aftersingle and repeated cutaneous applications for 4 weeks, on an “atopic”adult volunteer with very dry and squamous body skin.

1.2 Relevance of the Test

“Double-blind” evaluations, based on:

-   -   the principles of electrical conductivity of the skin, widely        described for determining the state of moisturization of the        upper layers of the epidermis (Tagami H. et al., 1980; Korstanje        et al., 1992);    -   the principle of photometry (sebumetric measurements), for        evaluating the relipidizing effect of a cosmetic product;    -   analysis by optical microscopy of surface “biopsies”, performed        by stripping, using cyanoacrylate adhesive, making it possible        to determine the effect of a cosmetic product on the        “MicroDepression Network” (MDN);    -   a clinical evaluation by the Study Director, a self-evaluation        by the panelist and a questionnaire.

1.3 Inclusion Criteria

“Atopic” volunteer with dry to very dry and slightly squamous body skin(dryness score ≧5 on a scale from 1 to 9).

1.4 Population Studied

18 “atopic” adult female volunteers (or 20 for the kinetics), from 20 to28 years old, with dry or very dry skin (2 dropouts from the study, notassociated with the applications).

1.5 Modes of Application

Single application: 0.07 ml of product, i.e. 2 μl/cm2, on one or twoareas of about 35 cm2 delimited on the skin of the right or left leg,according to a chance randomization. A control area was also delimitedfor each measurement type (corneometry and sebumetry).

Repeated uses: twice a day, under the normal conditions for use, for 4consecutive weeks, by the volunteer himself at his home, to half thebody.

1.6 Methodology

Single application: measurement of the electrical capacitance using aCornéométre™ (Courage+Khazaka electronic GmbH, Germany) and of theinitial level of surface skin lipids using a Sébumètre™ SM 810 PC(Courage and Khazaka) on areas treated with the test product(moisturization kinetics only), and also on an untreated control area(one control area per measurement type) before and then 1, 2, 3 and 24hours approximately after applying the products.

Repeated uses:

-   -   measurement of the electrical capacitance using a Cornéomètre™        (Courage+Khazaka electronic GmbH, Germany) on areas treated with        the test product before, and then after the 4 weeks of use of        the product;    -   production of surface “biopsies” by stripping, followed by        analysis by optical microscopy, on semi-structured linear scales        of 12 cm, taking into account the cleanliness of the        Microdepression Network and the surface appearance, before and        then after the 4 weeks of applications;    -   clinical evaluation by the Study Investigator and        self-evaluation by the panelists of the “dryness”, the        “roughness” and the “desquamation” of the skin, on the basis of        clinical scores or of analogue visual scales, at the same times        as previously;    -   assessment of the local skin tolerance of the product by the        Dermatologist, after the 4 weeks of use;    -   temperature and relative humidity regulated and checked at each        testing time (T°=22±2° C. and RH=50±5%).

1.7 Statistics

Instrumental measurements (corneometry and sebumetry): ANOVA andmultiple-comparisons test (p<0.05) relating to the absolute values andto the differences (Δ Tx−T0).

MDN, analogue scales and clinical scores: Wilcoxon test in paired series(“two-tail”, p<0.05).

Calculation of the percentages of variation of the parameters evaluatedduring the study.

2) Results

2.1 Effect on the Level of Surface Skin Lipids after Single Application(Sébumètre™)

A statistically significant increase in the level of surface skin lipidsrelative to the initial measurements and to the values recorded on thecontrol area, 1 and then 2 and 3 hours approximately after the firstapplication, are found, reflecting an immediate marked relipidizingeffect, not revealed approximately 24 hours after the application(reflecting a total absorption of composition 1.1, without the presenceof a residual greasy film on the surface of the skin).

2.2 Effect on the Degree of Moisturization of the Upper Layers of theEpidermis after Single and Repeated Applications (Cornéomètre™)

-   -   After single application (n=20)

A statistically significant increase in the electrical capacitance isfound relative to the initial measurements and to the measurementsrecorded on the control area, 1 and then 2, 3 and 24 hours approximatelyafter the first application of composition 1.1.

-   -   After 4 weeks of repeated use (n=18)

A statistically significant increase in the electrical capacitancerelative to the initial measurements is found.

TABLE 5.1 GAINS IN COMPOSITION 1.1 MOISTURIZATION Control area(EXAMPLE 1) T 1 hour +0.0% +48.6%° T 2 hours +1.0% +58.7%° T 3 hours+0.5% +64.4%° T 24 hours +2.9% +34.2%° T 4 weeks +22.4%° °statisticallysignificant increase in comparison with the untreated control area

2.3 Effect on the Microdepression Network

(analysis by optical microscopy of surface “biopsies”: 12 cmsemi-structured linear scales)

A statistically significant restructuring of the MicroDepression Networkis found, after 4 weeks of application.

TABLE 5.2 COMPOSITION 1.1 (EXAMPLE 1) Microrelief +10% Surfaceappearance +52%

2.4 Clinical Evaluation by the Study Director

(g-point clinical scores)

A statistically significant variation in the following judgementcriteria is observed, after 4 weeks of application:

TABLE 5.3 COMPOSITION 1.1 (EXAMPLE 1) Skin dryness −54% (p = 0.0002)Skin roughness −52% (p = 0.0002) Desquamation −54% (p = 0.0004)

2.5 Self-Evaluation by the Volunteers

(10-point analogue visual scales)

A statistically significant variation in the following judgementcriteria is observed, after 4 weeks of application:

TABLE 5.4 COMPOSITION 1.1 (EXAMPLE 1) Skin dryness −60% (p = 0.0002)Skin roughness −55% (p = 0.0002) Desquamation −60% (p = 0.0002)

3) Conclusion

. In conclusion, the single application to the skin of composition 1.1to 20 “atopic” female adult volunteers with dry to very dry skin, incomparison with an untreated control area (under double-blindconditions), resulted in:

-   -   a statistically significant effect on the content of surface        skin lipids (photometric measurements), in comparison with an        untreated control area, reflecting a marked immediate        relipidizing effect, not revealed 24 hours approximately after        the application, reflecting a total absorption of the product,        without the presence of a residual greasy film on the surface of        the skin;    -   a marked effect on the degree of moisturization of the upper        layers of the epidermis (electrical capacitance measurements),        1, 2, 3 and 24 hours approximately after the application,        reflecting an excellent remanence.

The repeated applications, twice a day for 4 consecutive weeks, underthe normal conditions for use, by a panel of 18 adult femaleindividuals, moreover, resulted in:

-   -   a statistically significant effect on the degree of        moisturization of the upper layers of the epidermis;    -   a statistically significant restructuring of the microdepression        network;    -   a statistically significant improvement in the appearance of the        skin (dryness, roughness and desquamation).

A positive judgement was also given by the majority of the panelists forthe efficacy of composition 1.1 as a “care cream for dry skin”, and alsofor its cosmetic qualities.

The applications of the studied composition 1.1 were found, moreover, tobe very well tolerated.

This set of results thus makes it possible to justify, for composition1.1, the following properties:

-   -   an immediate relipidizing effect,    -   an immediate and long lasting moisturizing effect on the upper        layers of the epidermis,    -   an improvement in the appearance of the skin, and    -   a tolerance and efficacy tested under dermatological monitoring.

1. A method of treating sensitive skin, irritated skin, reactive skin,atopic skin, pruritus, ichtyosis, acne, xerosis, atopic dermatitis,cutaneous desquamation, skin subjected to actinic radiation, or skinsubjected to ultraviolet radiation, comprising administering aneffective amount of a composition comprising furan lipids of plant oilthereby; and thereby increasing synthesis of skin lipids.
 2. The methodof claim 1, wherein furan lipids of plant oil are furan lipids ofavocado.
 3. The method of claim 1, wherein the composition isadministered topically.
 4. The method of claim 3, wherein thecomposition is applied to skin, the neighboring mucous membranes and/orinteguments thereof.
 5. The method of claim 1, wherein the compositionis administered orally.